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WO1996005287A1 - Procede permettant d'isoler des lymphocytes t du sang peripherique - Google Patents

Procede permettant d'isoler des lymphocytes t du sang peripherique Download PDF

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Publication number
WO1996005287A1
WO1996005287A1 PCT/US1995/010048 US9510048W WO9605287A1 WO 1996005287 A1 WO1996005287 A1 WO 1996005287A1 US 9510048 W US9510048 W US 9510048W WO 9605287 A1 WO9605287 A1 WO 9605287A1
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cells
mhc
peptide
complex
die
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PCT/US1995/010048
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English (en)
Inventor
Teresa Kendrick
Bishwajit Nag
Prabha V. Mukku
Somesh D. Sharma
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Anergen, Inc.
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Publication of WO1996005287A1 publication Critical patent/WO1996005287A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing

Definitions

  • This invention pertains to the field of immunology. More specifically, this invention provides a novel method of isolating pre-selected antigen-specific T- lymphocytes from a biological sample.
  • the isolation of T cells has been generally accomplished using lengthy procedures that involve a series of antigen stimulations in the presence of autologous antigen-presenting cells (APCs) which result in an increase in the clo ⁇ al frequency of the T cells specific for the particular peptide bearing APC.
  • APCs autologous antigen-presenting cells
  • the increased clonal frequency is a result of APC-induced proliferation of the T cell specific for the particular APC.
  • Limiting dilution is often used to select for the growth of the targeted cell (see, for example, pp 3.15.9-12 in Current Protocols in Immunology, Coligan et al. (eds) (1991).
  • the reliance on cell division to obtain a clonal population of cells makes this method a long and slow process requiring extensive labor and attention.
  • this invention also provides a method of detecting the presence, in a biological sample, of antigen presenting cells bearing an antigen by (a) providing T cells specific for an MHC:antigen complex; (b) contacting the biological sample with the T cells; and (c) detecting changes in the proliferation rate of said T cells.
  • This invention provides a novel and rapid method for isolating specific major histocompatibility complex (MHC): antigen-restricted T cells.
  • MHC major histocompatibility complex
  • this method does not rely on cell division or limiting dilution and provides far more rapid isolation requiring significantly less labor and attention.
  • the MHC restriction type may be dictated prior to the T cell isolation. It is desirable to isolate MHC:antigen-restricted T cells in a number of contexts.
  • adoptive immunotherapy for example, lymphocytes are removed from a patient, expanded ex vivo, and then re-infused back into the patient where they augment 3 the patient's native immune response (see U.S. Patent No.
  • MHC bound antigen can be detected by exposing the isolated T cells to a tissue sample (e.g., peripheral blood) and monitoring their proliferation rate.
  • tissue sample e.g., peripheral blood
  • isolation of MHC:antigen-restricted T cells provides a homogeneous source of T cell receptors.
  • a homogeneous source aids the elucidation of structure-function relationships of particular receptors. It also facilitates the development of solubilized T cell receptors which are of use in a number of therapeutic applications. (See, for example, PCT Patent Application No. 9201715 for Davis, et al. and U.S. Patent No. 5,283,058).
  • isolated T cell(s) refers T cells that are substantially or essentially free from components which normally accompany them as found in their native state. In particular the isolated T cells are essentially free from other T cells having a different antigen specificity or MHC restriction.
  • isolated T cells having a pre-selected antigen specificity and MHC restriction refers to the preferential selection and removal of T cells having an a priori determined particular antigen specificity and MHC restriction from a population of T cells that is heterogeneous in their antigen specificity and MHC restriction.
  • the T cell isolation method of this invention relies on the creation of a complex comprising an effective portion of one or more MHC encoded antigen- presenting glycoproteins bound to a peptide representing a fragment of an autoantigen or other antigenic sequence (i.e., an antigenic peptide).
  • This complex is referred to herein as an MHC:peptide complex.
  • Methods of forming MHC:peptide complexes are described by Sharma et al. Proc. Natl. Acad. Sci. USA, 88: 11465-11469 (1991) and in U.S. Patents 5,216,132 and 5,194,425.
  • the MHC:peptide complex is immobilized on a solid support and then exposed to a biological sample from which the T cells are to be isolated.
  • Specific binding T cells or T cells specific for a preselected MHC:peptide complex refers to those T cells in a heterogenous population of T cells which bind to a particular pre-deterrnined MHC:peptide complex with greater specificity and affinity than at least 80%, more preferably at least 90%, and still more preferably at least 95% of the T cells in the population.
  • Class I glycoproteins are found on the surfaces of all cells and are primarily recognized by cytotoxic T cells, while Class ⁇ glycoproteins are found on the surface of several cells, including accessory cells such as macrophages, and are involved in the presentation of antigens to T helper cells.
  • MHC proteins have been isolated and characterized. For a general review of MHC glycoprotein structure and function, see, e.g., Fundamental Immunology (2d Ed., W.E. Paul, (ed.), Ravens Press, N.Y. (1993)), which is incorporated herein by reference.
  • MHC component refers to one or more MHC glycoproteins or an effective portion of one or more MHC glycoproteins (i.e. , comprising an antigen binding site or sites and the sequences necessary for recognition by the appropriate T cell receptor) which is in other than its native state (i.e., not associated with the cell membrane of the cell that normally expresses MHC).
  • the MHC component is preferably solubilized from an appropriate cell source.
  • human lymphoblastoid cells are particularly preferred as sources for the MHC component.
  • the MHC glycoprotein portions of the complexes of the invention can be obtained by isolation from lymphocytes and screened for their ability to bind the desired peptide antigen.
  • the lymphocytes are preferably obtained from the species from which it is desired to isolate MHC:antigen-specific T cells.
  • the MHC-encoded glycoprotein component will preferably be obtained from human B cells which have been immortalized by transformation with a replication deficient Epstein-Barr virus, utilizing techniques known to those in the art.
  • MHC glycoproteins have been isolated from a multiplicity of cells using a variety of techniques including, for example, solubilization by treatment with papain, by treatment with 3M KC1 and by treatment with detergent.
  • solubilization by treatment with papain
  • 3M KC1 by treatment with 3M KC1
  • detergent extraction of Class ⁇ protein from lymphocytes followed by affinity purification is used.
  • the detergent can subsequently be removed by dialysis or through the use of selective binding beads, e.g. , Bio Beads.
  • the isolated antigens encoded by the I-A and I-E subregions have been shown to consist of two noncovalently bonded peptide chains: an alpha chain of 32-38 kD and a beta chain of 26-29 kD.
  • a third, invariant, 31 kD peptide is noncovalently associated with these two peptides, but it is not polymorphic and does not appear to be a component of the antigens on the cell surface (Sekaly, J. Exp. Med. 164:1490-1504
  • HLA human Class I histocompatibility proteins
  • the MHC of humans (HLA) on chromosome 6 has three loci, HLA-A, HLA-B, and HLA-C, the first two of which have a large number of alleles encoding alloantigens. These are found to consist of a 44 kD subunit and a 12 kD beta 2 -microglobuIin subunit which is common to all antigenic specificities. Isolation of these detergent-soluble HLA antigens was described by Springer, et al., Proc. Natl. Acad. Sci.
  • MHC glycoproteins since the amino acid sequences of a number of MHC glycoproteins are known and the genes have been cloned, one may express the desired MHC glycoprotein in a recombinantly engineered cell such as, for example, bacteria, yeast, insect (especially employing baculoviral vectors), and mammalian cells using conventional techniques know to those of skill in the art. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of the DNA encoding the MHC glycoproteins. In brief, the expression of natural or synthetic nucleic acids encoding
  • the transformed cells are then cultured under conditions favoring expression of the MHC sequence and the recombinantly produced protein recovered from the culture.
  • the recombinant MHC protein can be purified using conventional techniques known to those of skill in the art.
  • a metal binding domain e.g. , a sequence encoding a polyhistidine sequence
  • a metal binding domain e.g. , a sequence encoding a polyhistidine sequence
  • the recombinantly produced MHC polypeptides containing the metal binding domain can be purified using metal chelate chromatography. (See, section pertaining to "Metal Chelate Affinity Chromatography," infra.)
  • T cell epitopes have been identified in honey bee venom allergens, dust mite allergens, and ragweed allergens.
  • T cell epitopes have been identified in a number of infectious diseases including, for example, tetanus toxoid and pertussis toxin.
  • autoimmune diseases A large number of antigenic proteins or tissues for autoimmune diseases are also well known.
  • native type-II collagen has been identified in collagen-induced arthritis in rat and mouse, and mycobacterial heat shock protein in adjuvant arthritis (Stuart, et al. , (1984), Ann. Rev. Immunol. 2:199-218; van Eden, et al., (1988), Nature 331:171-173.); thyroglobulin has been identified in experimental allergic thyroiditis (EAT) in mouse (Maron, et al., (1988), J. Exp. Med.
  • EAT experimental allergic thyroiditis
  • acetyl choline receptor has been identified in experimental allergic myasthenia gravis (EAMG) (Lindstrom, et al. (1988), Adv. Immunol. 42:233-284); and myelin basic protein (MBP) and proteolipid protein (PLP) have been identified in experimental allergic encephalomyelitis (EAE) in mouse and rat (See Acha-Orbea, et al., supra).
  • target antigens have been identified in humans: type-II collagen has been identified in human rheumatoid arthritis (Holoshitz, et al.
  • a set of labeled test peptides is prepared and those which bind to an MHC in planar lipid membranes containing MHC proteins are shown to contain the agretope.
  • the identified agretope- bearing peptides are then prepared by conventional solid phase synthesis, and the subset which also contains epitopes for the disease-inducing T-helper cell clones is determined by incubation of the candidate peptides with murine antigen-presenting cells (APC) (or with isolated MHC complex) and spleen or lymph node T cells from mice immunized with the full length protein. Successful candidates will stimulate T cell proliferation in this system.
  • APC murine antigen-presenting cells
  • This second, smaller subset represents the suitable peptide component.
  • the peptides carrying agretopes permitting presentation of the epitopes associated with the alpha subunit of this receptor are readily determined. For example, determination of the appropriate peptides in a mouse model is carried out as follows.
  • mice which when immunized with Torpedo californicus AChR develop a disease with many of the features of human myasthenia gravis, are used as the model.
  • MHC Class ⁇ glycoproteins are isolated from spleen cells of these mice using lectin and monoclonal antibody affinity supports. The purified MHC Class ⁇ proteins are incorporated into phospholipid vesicles by detergent dialysis. The resultant vesicles are then allowed to fuse to clean glass cover slips to produce on each cover slip a planar lipid bilayer containing MHC molecules (See, Brian and McConnell, Proc. Natl. Acad. Sci. USA (1984) 81:6159, which is incorporated herein by reference).
  • the bound peptide contains an agretope (MHC Class II peptide binding site) of one of the several species of MHC Class II molecules present in the planar lipid membrane.
  • agretope MHC Class II peptide binding site
  • the set of agretopes for the alpha subunit of AChR is defined for the mouse strain that displays the symptoms of MG upon immunization with AChR or purified alpha subunit.
  • each of the alpha subunit synthetic peptide segments that contain an agretope is again incorporated into the antigen binding site of isolated MHC Class ⁇ proteins embedded in planar lipid membranes on cover slips.
  • the Dupont apparatus and technique for rapid multiple peptide synthesis are used to synthesize the members of a set of overlapping (10 residue overlap), 20-residue peptides from the alpha subunit of Torpedo califomicus AChR.
  • the sequence of this peptide is known.
  • One or more radioactive amino acids is incorporated into each synthetic peptide.
  • the pentafluorophenyl active esters of side chain-protected, FMOC amino acids are used to synthesize the peptides, applying standard stepwise solid phase peptide synthetic methods, followed by standard side chain deprotection and simultaneous release of the peptide amide from the solid support.
  • the overlapping sequences which include the putative segments of 8-18 amino acids of the antigenic protein, such as acetylcholine receptor protein can be synthesized using the method of Geysen, et al. , J. Immun. Meth. (1987) 102:274, which is incorporated herein by reference.
  • the synthesized radiolabeled peptides are subsequently tested by incubating them individually (on the plates) with purified MHC proteins which have been formulated into lipid membrane bilayers as described above.
  • MBP myelin basic protein
  • EAG experimental allergic encephalitis
  • the relevant antigenic sequences are relatively short in length (typically less than 25 amino acids), they can be readily synthesized using standard automated methods for peptide synthesis. In doing so, at least one metal-chelating amino acid is incorporated at either the N- or the C-terminus of the protein. Preferably, from two to about ten metal-chelating amino acids are incorporated into the antigenic peptide. More preferably, about six metal-chelating amino acids are incorporated into the antigenic peptide.
  • the antigenic peptide tagged with at least one metal- chelating amino acid can be made recombinantly using isolated or synthetic DNA sequences.
  • MHC-Peptide Complex Formation of the MHC-Peptide Complex.
  • the antigenic peptides can be associated noncovalently with the pocket portion of the MHC protein by, for example, mixing the two components together. This is typically done in an aqueous buffer that preserves the native conformation of the respective molecules.
  • Methods of producing MHC:peptide complexes are described by Sharma et al. Proc. Natl. Acad. Sci. USA, 88: 11465-11469 (1991) and in U.S. Patents 5,216,132 and 5,194,425.
  • excess peptide can be removed using a number of standard procedures, such as, for example, by ultrafiltrati ⁇ n or by dialysis.
  • MHC:peptide complexes may be purified by a number of means known to those of skill in the art.
  • the MHOpeptide complex may be isolated using a biotin-avidin system where the antigenic peptide contains a long chain thiol cleavable biotin moiety.
  • metal chelate affinity chromatography takes advantage of the reversible interaction between metal ions (such as, for example, Cu 2+ , Zn 2+ , Ni 2+ , etc.) and electron donor groups situated on the surface of proteins, especially the imidazole side-chain of histidine.
  • metal ions such as, for example, Cu 2+ , Zn 2+ , Ni 2+ , etc.
  • electron donor groups situated on the surface of proteins, especially the imidazole side-chain of histidine.
  • a chelating ligand i.e. , by forming a chelate resin
  • the protein binds to the immobilized metal ions when the pH is such that the electron donor group is at least partially unprotonized.
  • the bound protein can subsequently be eluted using a number of different techniques such as, for example, by competitive elution, by lowering the pH or, by using strong chelating agents.
  • Metal ions suitable for use in accordance with the present purification method include, but are not limited to, the first-row transitions metals.
  • the first-row transition metals include, for example, Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ and Fe 3 *.
  • Ni 2+ , Cu 2+ and Zn 2+ are the metal ions used. It has been found that these metals readily form a complex or chelate with metal-chelating amino acids and thus, they can be used to separate those proteins having a metal-chelating amino acid(s) from those which do not.
  • Metal-chelating amino acids suitable for use in accordance with the present method include those that are capable of participating in metal binding, i.e. , those amino acids that are capable of forming a chelate or complex with a metal ion.
  • Metal-chelating amino acids include, but are not limited to, the following: glycine, tyrosine, cysteine, histidine, arginine, lysine, asparagine and methionine.
  • histidine is the metal-chelating amino acid incorporated into the antigenic peptide. Since histidine is a relatively rare amino acid, accounting for only about 2% of the amino acids in most globular proteins (Klapper, M.H., Biochem. Biophys. Res.
  • selective separation of histidine-tagged polypeptides from a complex mixture can be achieved under both native and denatured conditions.
  • from two to about ten metal-chelating amino acids are incorporated into the antigenic peptide using standard automated methods for peptide synthesis.
  • about six metal-chelating amino acids are incorporated into the antigenic peptide.
  • Chelating ligands suitable for use in accordance with the present method include, but are not limited to, the following: iminodiace ⁇ c acid (“IDA”), N,N,N'-tris(carboxymethyl)ethylenediamine (“TED”), N-carboxymethyl aspartate, N,N,N,N,N-carboxymethyl tetraethylenepentamine and nitrilotriacetic acid (“NTA”).
  • IDA iminodiace ⁇ c acid
  • TED N,N,N'-tris(carboxymethyl)ethylenediamine
  • NTA nitrilotriacetic acid
  • nitrilotriacetic acid When charged with Ni 2+ , nitrilotriacetic acid is especially useful for the purification of proteins containing neighboring histidine residues.
  • the use of nitrilotriacetic acid as a chelating ligand is described in U.S. Patent No. 5,047,513, which is incorporated herein by reference.
  • the chelating ligand is covalently bound to a solid support or, alternatively, a chelating matrix using conventional methods and techniques known to and understood by those skilled in the art.
  • the solid support, having the chelating ligand bound thereto, is subsequently charged with a metal ion.
  • the solid support employed is not a critical aspect of the present invention.
  • Materials suitable for use as the solid support include those materials commonly used in affinity and gel chromatography. Examples of such materials include, but are not limited to, the following: dextran, agarose, cellulose, polystyrene, polyacrylamide, and their derivatives.
  • agarose or a derivative thereof (such as, for example, SepharoseTM (Pharmacia Biosystems, Uppsala, Sweden)) is used as the solid support.
  • the solid support charged with the metal ion, can be used batch-wise or in a chromatography column.
  • the solid support is packed into a column (e.g., 14.5 cm x 1.6 cm), and equilibrated with an aqueous buffer that does not form chelates with the metal ion employed. It will be readily apparent to those skilled in the art that the dimensions of the column can be varied depending upon the quantity of protein to be purified.
  • Equilibration buffers suitable for use in the present method include, for example, a sodium or potassium phosphate buffer (pH 7- 8) or a Tris-HCl buffer (pH 7.0).
  • the ' 1 6 solid support is then washed with equilibrating buffer to remove the uncomplexed MHC molecules and other MHC-peptide complexes, neither of which will bind to the solid support due to the absence of a metal-chelating amino acid.
  • the solid support can be washed with a number of different reagents to remove residual contaminations due to disulfide cross links, hydrophobic interactions, low affinity binding to the resin, etc.
  • the equilibrating buffer can contain a denaturing agent or a detergent, such as, for example, guanidine, NaCl, ethanol, glycerol, urea, Tween or TritonTM.
  • the bound MHC-peptide complexes of interest are eluted from the column by washing the solid support with an elution buffer.
  • the elution buffer can be of a constant pH or can be applied as a pH gradient.
  • the elution buffer can contain either a Lewis acid, i.e. , an electron acceptor, which competes with the metal for the protein, or a Lewis base, i.e., an electron donor, which competes with the protein for the metal.
  • the elution buffer used is 0.05 M imidazole; imidazole will compete with the protein for the metal coordination sites thereby displacing the MHC-peptide.
  • the optimal elution conditions are dependent on the amount and type of impurities present, the amount of material to be purified, the column dimensions, etc. Such conditions are readily determined on a case by case basis by those of ordinary skill in the art.
  • the isolated MHC:peptide complex is immobilized by attaching it to a solid support.
  • a solid support is well known to those of skill in the art.
  • a suitable support is one which provides a functional group for the attachment of the MHC:peptide complex either directly or indirectly through a linker.
  • a solid support comprises a solid phase material deriva ⁇ zed with functional groups to facilitate the chemical coupling of the MHC:peptide complex or a linker joined to the MHC:peptide complex.
  • the solid support should be inert to the reagents to which it will be exposed.
  • Suitable solid support materials include, but are not limited to, polacryloylmorpholide, silica, controlled pore glass (CPG), polystyrene, polystyrene/latex, carboxyl modified teflon, derivatized magnetic beads, and even glass or polymer cell culture dishes and die like.
  • the MHC:peptide complexes may be immobilized on the solid support either directly through a covalent bond (i.e., through the terminal carboxyl or amino groups of the MHC-encoded glycoprotein) or indirectly through one or more linkers.
  • linker refers to a single molecule or a complex of molecules that join die MHC:peptide complex to the solid support.
  • linker may be joined to the MHC:peptide complex, the solid support, or to other linker molecules through covalent bonds, ionic bonds, van der Waals interactions, or hydrophobic interactions either individually or in combination.
  • Linkers suitable for joining peptides are well known to those of skill in the art. Generally linkers are either hetero- or homo-bifunctional molecules that contain two reactive sites that may form a covalent bond with the
  • MHC:peptide complex and with the solid support respectively.
  • linker molecules are well known to those of skill in the art.
  • MHC:peptide complex may be joined to the solid support by a peptide linker, by a straight or branched chain carbon chain linker, or by a heterocyclic carbon linker.
  • Heterobifunctional cross linking reagents are well known to those of skill in the art. See, for example, Lemer et al. Proc. Nat'l. Acad. Sci. USA, 78: 3403-3407 (1981) and Kitagawa et al. J. Biochem., 79: 233-236 (1976) which arc incorporated herein by reference.
  • the linker comprises a first monoclonal antibody chemically conjugated to the solid support, where the first monoclonal antibody is specific to a component of a second monoclonal antibody which is, in turn, specific for a component of the MHC:peptide complex.
  • first monoclonal antibody is specific to a component of a second monoclonal antibody which is, in turn, specific for a component of the MHC:peptide complex.
  • magnetic beads may be chemically conjugated with a rat anti-mouse IgG (e.g. IgGzJ which, in turn, binds to mouse anti-human MHC antibody which binds to a human MHC:peptide complex.
  • a biological sample containing T cells is incubated with the solid support-bound MHC:peptide complex.
  • a "biological sample” refers to any sample obtained from a living organism or from an organism that has died. Examples of biological samples include body fluids and tissue specimens. In the case of microorganisms a biological sample may include samples containing many entire organisms.
  • T cells present in the biological sample that are specific for the MHC component and the bound peptide will bind to the MHC:peptide complex forming a 1 6
  • MHC:peptide:T cell complex The MHC:peptide:T cell complex is then removed from biological sample. This may be accomplished by simply washing away the biological sample while retaining the solid support. Alternatively, d e solid support bearing the MHC:peptide:T cell complex may be removed from the biological sample. In a preferred embodiment, where the solid support is a magnetic bead, the separation may be accomplished magnetically.
  • the MHC.'peptide-specific T cells are separate from the spontaneously from the complex when it is incubated overnight at 37°C.
  • the isolated T cells may then be grown in standard culture media (e.g. RPMI 1640, 10% fetal bovine serum, 2mM L- glutamine, 20 U/ml recombinant human interleukin-2).
  • Interleukin-2 is added in order to reverse potential anergy due to lack of T cell costimulation via B7(APC)-CD28(T cell) interactions.
  • the T cells may be challenged, in culture, with the particular peptide fragment that was bound to the MHC- encoded glycoprotein and autologous antigen presenting cells (APCs). If the T cells are specific for the particular peptide MHC class present on the APCs, they will proliferate in response to the challenge. T cell proliferation may be measured by a number of means well known to those of skill in die art. Typical metiiods involve either measuring die increase in cell number or alternatively, measuring changes in metabolic rate as determined by d e rate of uptake of a labeled metabolic substrate.
  • [ 3 H]-tt ⁇ ymidine is preferred.
  • the T cells are specific to die particular peptide and MHC class, they will show a MHC/peptide dose-dependent increase in the rate of [ 3 H]-thymidine uptake.
  • HLA DR2 HLA DRB1*1501 and DRB5*0101
  • myelin basic protein peptide analog His 77'82 Y 83 MBP84-102
  • HLA DR2 refers to the DR2 glycoproteins of the human MHC complex.
  • the myelin basic protein analog His 7M2 Y u MBP84-102 is a peptide containing six histidines at the amino terminus followed by a tyrosine and men residues 84 through 102 of die myelin basic protein.
  • the specifically loaded complexes, designated DR2:MBP84-102 were purified from uncomplexed molecules or complexes with nonspecifically bound peptides by metal chelate affinity chromatography as described by Nag et al. Id.
  • the DR2:MBP84-102 complex was then immobilized onto magnetic beads. This was accomplished by incubating magnetic beads conjugated to rat anti-mouse IgG j , (Dynal, Inc. Great Neck, N.Y., USA)) with mouse anti-human MHC class ⁇ DR monoclonal antibody, L243 (ATCC Deposit Number HB55) and DR2:MBP84-102 at 37 ⁇ C for 1 hour in a phosphate buffer solution (0.1 M NaPO4, pH 7.5, 0.1 % human serum albumin, 0.02% NaN 3 ). The beads, now bearing the DR2:MBP84-102 complex were then washed four times in RPMI 1640 medium (Gibco-BRL, Grand Island New York, USA).
  • the magnetic bead:DR2:MBP84-102 complexes were incubated witii donor cells for 20 minutes on ice in RPMI 1640 supplemented witii 10% fetal bovine serum, 2 ⁇ M L-glutamine, and 10 ⁇ g/ml phytohemaglutinin. Beads with cells attached were separated magnetically, washed four times with die incubation medium and then maintained in incubation medium supplemented witii 20 U/ml rIl-2.
  • T cells were challenged witii MBP84-102 and autologous APCs.
  • T cells were cultured witii 5 x 10 s freshly irradiated autologous APCs (peripheral blood lymphocytes) in d e presence of increasing concentrations of MBP84-102.
  • autologous APCs peripheral blood lymphocytes
  • 1 ⁇ Ci of [ 3 H]-thymidine was added and the degree of proliferation was measured by incorporated radioactivity.
  • the T cell proliferation rate showed a dose dependent response to MBP84-102 and autologous APCs indicating die presence of T cells specific to MBP84-102 ( Figure 1).
  • the precursor cell frequency in a normal individual is approximately one in a million.
  • the magnitude of response following an initial stimulation/antigen-specific proliferation assay is too great to be accounted for by differential proliferation of die DR2 specific T cells without actual isolation and enrichment of the population for those cells.
  • T cells presumably specific to MBP(84-102) were isolated as described in Example 1.
  • the isolated T cells were cultured in triplicate with 5 x 10 s freshly irradiated autologous APC cells in die presence of 0, 5, 10, and 20 ⁇ g/ml of tidier 6xHis-MBP(83-102)YH 13 peptide, MBP(84-102) or with irrelevant peptide MBP(124- 143).
  • the degree of T cell proliferation was measured by [ 3 H]-thymidine incorporation.
  • FIG. 2 shows d e antigen specificity of the isolated T cells.
  • the cells proliferated in the presence of peptides containing die epitope MBP(84-102), yet failed to proliferate in the presence of anodier epitope MBP(124-143) from die same myelin basic protein under identical conditions.
  • d e claimed metiiod isolated MBP(84-102) specific T cells.

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Abstract

L'invention concerne un procédé nouveau et rapide qui permet d'isoler des lymphocytes T limités à des antigènes codés par le complexe majeur d'histocompatibilité. Ce procédé consiste à: (a) prendre un complexe déjà isolé, de peptides codés par le complexe majeur d'histocompatibilité, lié à un support solide; (b) mettre ce complexe en contact avec l'échantillon biologique contenant des lymphocytes T pour former un complexe lymphocytes T/peptides codés par le complexe majeur d'histocompatibilité; (c) extraire ce complexe lymphocytes T/peptides codés par le complexe majeur d'histocompatibilité du sang périphérique; et (d) séparer les lymphocytes T liés de ce complexe lymphocytes T/peptides codés par le complexe majeur d'histocompatibilité.
PCT/US1995/010048 1994-08-09 1995-08-09 Procede permettant d'isoler des lymphocytes t du sang peripherique WO1996005287A1 (fr)

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US08/288,143 US5595881A (en) 1994-08-09 1994-08-09 Method for the detection of antigen presenting cells

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EP0937251A1 (fr) * 1996-09-06 1999-08-25 Ortho Pharmaceutical Corporation Purification de cellules t specifiques de l'antigene
EP0972518A2 (fr) * 1998-07-10 2000-01-19 Universite Pierre Et Marie Curie Paris Vi Echange de lymphocytes T
EP0812331A4 (fr) * 1995-02-28 2000-06-07 Trustees Of Board Of Complexes antigenes/mhc pour detecter et purifier les lymphocytes t specifiques aux antigenes
WO2002054065A2 (fr) * 2000-12-28 2002-07-11 Iba Gmbh Coloration reversible aux multimeres cmh pour une purification fonctionnelle de lymphocytes t specifiques de l'antigene
WO2009010474A1 (fr) * 2007-07-13 2009-01-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Anticorps ou fragment de celui-ci reconnaissant l'avitagtm et ses utilisations
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EP0812331A4 (fr) * 1995-02-28 2000-06-07 Trustees Of Board Of Complexes antigenes/mhc pour detecter et purifier les lymphocytes t specifiques aux antigenes
EP1437366A1 (fr) * 1995-02-28 2004-07-14 The Board Of Trustees Of The Leland Stanford Junior University Complexes antigènes/MHC pour détecter et purifier les lymphocytes T spécifiques aux antigènes
WO1997044667A3 (fr) * 1996-05-21 1998-03-19 Pasteur Institut Methodes d'utilisation de complexes peptide/complexe majeur d'histocompatibilite pour obtenir ou purifier des cellules T antigene-specifiques et pour stimuler des cellules T
WO1997044667A2 (fr) * 1996-05-21 1997-11-27 Institut Pasteur Methodes d'utilisation de complexes peptide/complexe majeur d'histocompatibilite pour obtenir ou purifier des cellules t antigene-specifiques et pour stimuler des cellules t
EP0937251A1 (fr) * 1996-09-06 1999-08-25 Ortho Pharmaceutical Corporation Purification de cellules t specifiques de l'antigene
AU727541B2 (en) * 1996-09-06 2000-12-14 Ortho-Mcneil Pharmaceutical, Inc. Purification of antigen-specific T cells
US7125964B2 (en) 1996-09-06 2006-10-24 Ortho-Mcneil Pharmaceutical, Inc. Purification of antigen-specific T cells
EP0937251A4 (fr) * 1996-09-06 2002-07-17 Ortho Mcneil Pharm Inc Purification de cellules t specifiques de l'antigene
EP0972518B1 (fr) * 1998-07-10 2004-11-24 Universite Pierre Et Marie Curie Paris Vi Echange de lymphocytes T
EP0972518A2 (fr) * 1998-07-10 2000-01-19 Universite Pierre Et Marie Curie Paris Vi Echange de lymphocytes T
WO2002054065A3 (fr) * 2000-12-28 2002-11-28 Iba Gmbh Coloration reversible aux multimeres cmh pour une purification fonctionnelle de lymphocytes t specifiques de l'antigene
WO2002054065A2 (fr) * 2000-12-28 2002-07-11 Iba Gmbh Coloration reversible aux multimeres cmh pour une purification fonctionnelle de lymphocytes t specifiques de l'antigene
US7776562B2 (en) 2000-12-28 2010-08-17 Iba Gmbh Reversible MHC multimer staining for functional purification of antigen-specific T cells
US8298782B2 (en) 2000-12-28 2012-10-30 Iba Gmbh Reversible MHC multimer staining for functional purification of antigen-specific T cells
US9222070B2 (en) 2001-02-20 2015-12-29 Janssen Pharmaceuticals, Inc. Cell therapy method for the treatment of tumors
US9222071B2 (en) 2001-02-20 2015-12-29 Janssen Pharmaceuticals, Inc. Cell therapy method for the treatment of tumors
WO2009010474A1 (fr) * 2007-07-13 2009-01-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Anticorps ou fragment de celui-ci reconnaissant l'avitagtm et ses utilisations
US8431394B2 (en) 2007-07-13 2013-04-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Antibody or fragment thereof recognizing Avitag™ and uses thereof

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